Prokaryotic collagen-like proteins and uses thereof

ABSTRACT

The present invention provides recombinant triple helical proteins or collagen-like proteins comprising a prokaryotic protein or one or more domains of a prokaryotic protein comprising a collagen-like peptide sequence of repeated Gly-Xaa-Yaa triplets and, optionally, one or more domains from a mammalian collagen. Also provided are expression vectors and host cells containing the expression vectors to produce these recombinant proteins and methods of production for the same. Additionally, antibodies are provided that are directed against a recombinant collagen-like protein that, preferably, binds an integrin. Furthermore, a method of screening for potential therapeutic compounds that inhibit the integrin-binding or -interacting activities of recombinant collagen-like proteins.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation-in-part application which claims the benefit ofpriority under 35 U.S.C. §120 of U.S. Ser. No. 10/135,795, filed Apr.30, 2002, which is a continuation-in-part application which claims thebenefit of priority under 35 U.S.C. §120 of U.S. Ser. No. 10/102,283,filed Mar. 20, 2002, now U.S Pat. No. 6,875,609, which is acontinuation-in-part application of U.S. Ser. No. 09/919,048, filed Jul.30, 2001, now U.S. Pat. No. 6,787,354, which is a continuation-in-partapplication of application Ser. No. 09/861,966, filed May 21, 2001, nowU.S. Pat. No. 6,518,028, which is a divisional application ofapplication Ser. No. 09/510,738, filed Feb. 22, 2000, now U.S. Pat. No.6,268,165, which claims the benefit of priority under 35 USC §120 ofU.S. Ser. No. 09/039,211, filed Mar. 14, 1998, which claims benefit ofprovisional patent application U.S. Ser. No. 60/041,404, filed Mar. 19,1997, now abandoned.

FEDERAL FUNDING LEGEND

This invention was produced in part using funds obtained through grantsAR44415 and AI50666 from the National Institutes of Health.Consequently, the federal government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to protein molecular biology andmicrobiology. More specifically, the present invention relates torecombinant prokaryotic collagen-like or triple helical proteins andmethods of producing collagen-like materials in prokaryotic organisms.

2. Description of the Related Art

Collagens are abundant extracellular matrix proteins that are essentialstructural elements of connective tissues in human and animals. Thevertebrate collagens are classified into 19 types that are grouped intwo major categories known as fibrillar, i.e., types I–III, V, and XIand nonfibrillar, i.e., types IV, VI–X, and XII–XIX collagens. Inaddition, collagens can alter cell function by interacting with specificcellular receptors (1). The repeating sequence Gly-Xaa-Yaa (GXY), inwhich a sterically small glycine residue occupies every third position,because only glycine is small enough to be accommodated at the center ofthe triple helix and in which X is often occupied by proline and Y byhydroxyproline, is a unique feature of the collagen polypeptide (2–4).Proline hydroxylation together with glycosylation of the polypeptide,are essential factors in stabilizing the collagen triple helix and theformation of collagen networks, respectively.

Long tracks of repeated GXY sequences fold into left-handed polyprolinetype II-like chains and three such chains cooperatively twist around acentral axis to form a right-handed rope-like superhelix (2,5–7). Thethree chains are linked by hydrogen bonds between the backbone group,—NH (Gly) and the backbone carbonyl group of residue X of another chain,and by water-mediated interactions. In humans, mutations that affectcollagen triple helix formation and fiber assembly have seriouspathological consequences often leading to death.

In some collagen types, the C-terminal and N-terminal propeptides areremoved during secretion from cells (7–9). The resulting mature collagenmolecules are deposited in the extracellular matrix in the form offibers, networks and beaded filaments (8). One group of mammalianproteins has collageneous subdomains, but they are not conventionalcollagens. This group includes several proteins that fulfill rudimentaryhost defense functions, including complement factor C1q (10) and somemammalian lectins (11). These proteins form characteristic lollipop-likestructures with stalks made from their collagenous domains and globularheads made from the non-collagenous regions.

Collagen-like molecules also have been found in lower eukaryotes, suchas mussels, worms, and sponges (12), and collagen-like sequences havebeen deduced from analyses of the genomes of prokaryotes (13–16).Moreover, DNA tracks encoding collagen-like sequences have been found inthe genomes of bacteria and phages. However, these organisms appear tolack proline hydroxylases and since hydroxyproline in the past has beenconsidered a critical residue for triple helix formation, it is unclearif the prokaryotic GXY repeated motifs result in proteins that can formstable collagen-like triple helices. Recent studies of model syntheticpeptides demonstrate that a GXY sequence with certain so called “guest”residues other than proline and hydroxyproline in the X and Y positionsare capable of forming a stable triple helix. Furthermore, type Icollagen expressed in tobacco plants, although virtually unhydroxylated,does form a triple helix.

Collagens can act as cell adhesion substrates, organize the cytoskeletonand promote cellular contractility and motility by their ability tointeract with integrins and cellular adhesion receptors (17–19).Integrins are large glycoproteins and are expressed as αβ heterodimerson the cell surface (17,19–21). There are 14 distinct a subunits andeight β subunits in mammals that combine to form 24 known heterodimers(22–23).

There are four major subunits of integrin that act as collagenreceptors, including α₁β₁ and α₂β₁, which are the most widely expressed,and α₁₀β₁ and α₁₁β₁, which are more distinctly distributed (24–26).While α₁β₁ and α₂β₁ favor Col IV and Col I, respectively (27–28), eachof these heterodimers is known to bind to both types of collagens (30).Even though α₁β₁ and α₂β₁ integrins interact with several types ofcollagen proteins, they appear to possess distinct recognitionabilities. For example, α₁β₁ integrin can bind to type XIII collagen,whereas α₂b₁ integrin cannot (30).

The extracellular domains of integrins interact with extracellularmatrix (ECM) proteins in a metal ion-dependent manner (31). Recentstudies demonstrate that the so called I-domains of the α subunits ofα₁β₁, α₂β₁, α₁₀β₁, and α₁₁β₁ integrins mediate the interactions of theseECM receptors with collagens and control cell adhesion activity (32–34).The cytoplasmic segments of integrins interact with elements of thecytoskeleton and the signaling molecules, and can trigger intracellularsignaling pathways. For example, integrin ligation induces tyrosinephosphorylation of FAK, PYK2, p72SYK, ILK-1, CAS, paxillin, SRC/FYN, andShc (35–40). Furthermore, signaling events mediated by these moleculesare important in an array of biological processes, includingcell-migration, cell proliferation and differentiation, angiogenesis,and cancer cell metastasis (35, 38, 40).

Denatured collagen, gelatin, is widely used in the cosmetic andpharmacological industries, for example, as a pill coating or as astabilizer. Collagen is usually obtained from bovine skin or otheranimal products. Unfortunately, these animal protein products can becontaminated by viruses and prions, such as occurs in mad cow disease.Mammalian collagens have been shown to induce autoimmune diseases inanimal models. An artificial collagen product would be a desirablealternative to animal based collagen products.

The Streptococcal collagen-like proteins, Scl1 and Scl2, also known asSclA and SclB, are the best-characterized members of the prokaryoticfamily of collagen-like proteins (41–45). The two related proteinscontain long segments of repeated GXY sequences and are located on thecell surface of the human bacterial pathogen, Streptococcus pyogenes ora group A Streptococcus (GAS). The Scl1 and Scl2 proteins have a similarprimary structure, which allows for the assignment of four commondomains (42). The amino terminal signal sequence and carboxyl terminalcell-wall associated regions are conserved between Scl1 and Scl2,whereas the variable (V) and the collagen-like (CL) regions differsignificantly in length and primary sequence. In addition, Scl1, but notScl2, contains a linker (L) region between the collagen-like and thecell-wall regions, which is composed of highly conserved tandem repeats.This model was recently supported by an extensive genome-based sequenceanalysis that further established the presence of putative collagen-likedomains in prokaryotic proteins (16).

Group A Streptococcus (GAS) are extracellular pathogens that can attachto and invade various human cell types using cellular receptors such asCD44, CD46, and integrins (46–50). Productive adherence is the firststep required for pathogenic bacteria to colonize, invade, divide, andsecrete virulence factors (51). Integrins are normally located on thebasal side of polarized cells and, therefore, may not be immediatelyaccessible for the interactions. It has been postulated that localtrauma is required for streptococci to invade deeper anatomical sites. Afew distinct routes, through which S. pyogenes emigrate into theunderlying tissues, have been identified.

One model proposes that bacterial invasion is dependent on the presenceof M1 protein on the cell surface. In this mechanism, streptococciefficiently invaded HeLa (epithelial) cells by a zipper-like mechanismmediated by host cell microvilli and the resulting endocytosis wasaccompanied by actin polymerization (52). In a paracellular model, theM3-serotype GAS strain producing hyaluronic acid (HA) capsule interactswith CD44 to promote the formation of lamellipodia in keratinocytes in aRac-1-dependent manner. This event also disrupts intercellular cell-celladhesion junctions, thereby allowing the pathogen to emigrate onto thebaso-lateral surface of cultured keratinocytes (50).

More recently, a study demonstrated that human umbilical veinendothelial cells (HUVECs) directly uptake GAS strain expressing surfaceprotein Sfb1 through “caveolae” (53). A primary component of thesecaveolae is a membrane bound protein caveolin-1, whose functions aredependent on sphingolipids and cholesterol (54–55). Pre-treatment ofHUVECs with methyl-β-cyclodextrin and filipin, drugs that disruptcaveolae and membrane-microdomain by removing lipid moieties, abolishedinvasion of HUVECs by GAS (53). It was previously shown that integrinsassociate with caveolin-1 (56–58).

Three other streptococcal proteins have been reported to interact withvarious integrins. For example, a secreted cysteine protease (SpeB)variant that contains an RGD sequence motif and is expressed by theserotype M1 strains that cause invasive disease was shown to bindintegrins α_(v)β₃ and α_(11b)β₃ (48,59). In addition, two FN-bindingstreptococcal cell surface proteins, SfbI/F1 and M1, were shown to bindto α₅β₁ integrin via FN (52, 60–61).

Streptococcal cell wall structures that molecularly mimic components ofthe human body have long been postulated to be a factor inpostinfectious autoimmune disease such as rheumatic fever andpoststreptococcal glomerulonephritis. In addition, microbial infectionsare presumed to play a triggering role in several other autoimmunediseases. Interestingly, autoimmune diseases are often associated withelevated levels of anti-collagen antibodies present in patients' sera.Furthermore, autoimmune diseases can be induced in experimental animalstreated with collagen. The discovery that Scl proteins have structuralsimilarity to collagens and during infection could induce antibodiescross-reacting with host collagens adds another dimension to GAS-inducedautoimmunity.

The inventors have recognized a need in the art for improvements inmethods of producing a new source of collagen and in methods ofemploying prokaryotic GXY sequences in function- and structure-relatedstudies. Specifically, the prior art is deficient in prokaryotic-likecollagens and in determining their role in employing host cell-specificreceptors, e.g., the collagen-binding integrins. The present inventionfulfills this long-standing need and desire in the art.

SUMMARY OF THE INVENTION

The present invention is directed to a recombinant triple helicalprotein. This recombinant protein comprises a prokaryotic protein or oneor more domains of a prokaryotic protein comprising a collagen-likepeptide sequence of repeated Gly-Xaa-Yaa triplets having non-modifiedamino acids. This recombinant triple helical protein may furthercomprise one or more domains of a biologically active mammalian proteinor peptide fragment therefrom.

The present invention is directed to a related recombinant collagen-likeprotein. The recombinant collagen-like protein comprises one or moredomains having a peptide sequence of one or more of SEQ ID NOS: 1–42.

The present invention is directed also to a chimeric collagen-likeprotein. The collagen-like protein comprises one or more domains havinga peptide sequence of one or more of SEQ ID NOS: 1–42 and one or moredomains of a human collagen protein.

The present invention also is directed to an expression vectorcomprising a DNA encoding the recombinant triple helical protein. Thepresent invention also is directed to a related expression vectorcomprising a DNA encoding the recombinant collagen-like protein.

The present invention also is directed to a host cell comprising anexpression vector described herein.

The present invention is directed further to a method of producing atriple helical protein. The method comprises introducing into aprokaryotic host cell an expression vector comprising a DNA sequenceencoding the triple helical protein described herein, culturing the hostcell under conditions suitable to express the protein and isolating theexpressed protein to produce a triple helical protein. Additionally, themethod may comprise purifying the triple helical protein. Furthermore,the method may comprise heating the triple helical protein and producinga gelatin-like material therefrom. In a related method the expressionvector comprises DNA encoding a triple helical protein having one ormore domains having a peptide sequence of one or more of SEQ ID NOS:1–42.

The present invention is directed further to another related method ofproducing a collagen-like protein having one or more domains having apeptide sequence of one or more of SEQ ID NOS: 1–42. The methodcomprises those steps described supra for a triple helical protein.

The present invention is directed further still to a method of producinga gelatin-like material. The method comprises introducing into aprokaryotic host cell an expression vector comprising a DNA sequenceencoding a protein having one or more domains having a peptide sequenceof one or more of SEQ ID NOS: 1–42, and culturing the prokaryotic hostcell under conditions suitable to express the protein. The expressedprotein is heated to produce the gelatin-like material.

The present invention is directed further yet to an antibody directedagainst a recombinant streptococcal collagen-like (rScl) protein. TherScl protein binds to α₂β₁ integrin. Also, the present invention isdirected to a related antibody directed against p176 which binds α₂β₁integrin. The present invention is directed further to another relatedantibody directed against a domain in p176 that binds an α₂-I domain ofα₂β₁ integrin.

The present invention is directed further still to a method forscreening for compounds that inhibit streptococcal collagen-like (Scl)protein binding to or interaction with an integrin. The method comprisesmeasuring Scl binding to or interaction with the integrin in thepresence of a test compound and measuring Scl binding to or interactionwith the integrin in the absence of the test compound in a controlsample. Binding of the integrin to Scl in the presence of the testcompound is compared with binding in the control sample. A reduction inbinding of the integrin to Scl in the presence of the test compoundcompared to control correlates with inhibition of integrin binding to orinteraction with Scl by the test compound.

Other and further aspects, features, and advantages of the presentinvention will be apparent from the following description of thepresently preferred embodiments of the invention. These embodiments aregiven for the purpose of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A–1D are characterizations of the recombinant rScl proteins. FIG.1A shows a schematic representation of the Scl1.1 and Scl2.28 proteinsand the recombinant polypeptides (not to scale). Ss, signal sequence; V,variable region; CL, collagen-like region; L, linker region; WM, cellwall/membrane region. All recombinant proteins, P144 (SEQ ID NO: 47),P157 (SEQ ID NO: 48), P158 (SEQ ID NO: 49), and P163, contain Strep-tagII (NH₂-WSHPQFEK-COOH; SEQ ID NO: 45) at their carboxyl termini. FIG. 1Bshows the 15% SDS-PAGE analysis of purified recombinant proteins.Duplicated samples of P144, P157, and P158 were transferred onto thenitrocellulose membrane. Immunoblots were probed with theaffinity-purified antibody directed against the V region of the Scl1.1protein.

FIG. 1C shows a schematic representation of the recombinant P176 andP163 (not to scale) and FIG. 1D shows a Commassie stained 12% SDS-PAGEintegrity analysis of purified rScls. V, variable region; CL,collagen-like region; L, linker region; tag, Strep-tag II(NH₂-WSHPQFEK-COOH).

FIGS. 2A–2C demonstrate the triple helix formation by Scl1 and Scl2.FIGS. 2A–2B show oligomerization of the recombinant proteins.Heat-denatured (90° C.) or native (4° C.) P144, P157, and P163recombinant proteins were electrophoretically separated in 10–20%gradient gels without (FIG. 2A) or with (FIG. 2B) SDS and visualized bystaining. Migration of molecular mass standards is shown in SDS-PAGE.FIG. 2C demonstrates resistance to trypsin digestion. Samples ofheat-denatured or native P144 were digested with trypsin at 15° C.Samples were separated in 12% SDS-PAGE, and band intensities of proteinresistant to trypsin were estimated at different time points.

FIGS. 3A–3H depict circular dichroism spectra and thermal stability ofrecombinant rScl proteins. FIGS. 3A–3C are wavelength scans of P144,P157, and P163 before and after unfolding and refolding. Solid lines, CDspectra at 4° C. (P144 and P163) and 25° C. (P157) before unfolding;broken lines, CD spectra at 50° C. after unfolding; dotted lines, CDspectra at 4° C. (P144 and P163) and 25° C. (P157) after refolding.FIGS. 3D–3F show thermal unfolding and refolding profiles of P144, P157,and P163. CD was recorded at 220 nm with a temperature slope of 20°C./h. I, unfolding curve; II, refolding curve. A connecting line wasused to show the refolding curve of P157. The concentrations of thesamples were: P144, 8.1 μM; P157, 29.8 μM; and P163, 7.7 μM. FIG. 3Gshows the circular dichroism spectrum of the variable region of Scl1.Wavelength scan of P158 (10 μM) at 25° C. FIG. 3H shows the circulardichroism spectra of P176 before unfolding (4° C. and 25° C.), afterunfolding (50° C.) and refolding (4° C., renatured).

FIGS. 4A–4G show the organization of the Scl1 and Scl2 proteins asviewed by electron microscopy after rotary shadowing. FIG. 4A shows afield demonstrating lollipop-like structures, monomers and dimmers,adopted by P144 (Scl1.1). An example of a P144 monomer is indicated withan arrow and a dimer is identified with an arrowhead. FIG. 4B showsselected monomers with a two-domain structure shown at highermagnification. FIG. 4C shows selected dimers at higher magnificationdemonstrating head-to-head interactions. FIG. 4D shows a fielddemonstrating the formation of rod-shaped particles by P157corresponding to the collagen-like region of Scl1.1. FIG. 4E showsidentification of the globular domain of P144 using anti-V regionantibody. An example of an antibody bound to the globular domain of P144(Scl1.1) is indicated with an arrow and an unbound antibody is indicatedwith an arrowhead. FIG. 4F shows a lollipop-like structure of P163(Scl2.28). FIGS. 4A and 4D–4F are shown at the same magnification. Bars:50 nm. FIG. 4G is an electron micrograph of the rotary shadowed P176,demonstrating the formation of lollipop-like structures.

FIGS. 5A–5B are a triple-helical model of the collagenous domains ofScl1.41 (SEQ ID NO.: 15) and Scl2.28 (SEQ ID NO.: 43) and the amino acidsequence of P176. In FIG. 5A three polypeptide chains were modeled basedon the crystallographic determination of a collagen-like peptidePro-Pro-Gly. Selected residues are depicted as follows: Pro; ionizableresidues Lys and Arg,bold; stabilizing triplets GPR, GER, GPA, GDR, GKD,and GEK are marked with black dots. In FIG. 5B the amino acid sequenceof P176 (SEQ ID NO.: 44) is shown in black. Selected residues aredepicted as follows: collagen-like region (CL), underlined; variableregion (V), regular and upper case; Bold, linker region; Italic, Cellmembrane/membrane region; Strep-tag II (stag), italic and lower case.

FIGS. 6A–6D show the expression of integrins on the surface of MCR-5cells (FIG. 6A) and WI-38 cells (FIG. 6B) and demonstrate adhesion ofMRC-5 (FIG. 6C) and) WI-38 fibroblast cells (FIG. 6D) after permissiveadhesion was allowed to occur for 45 or 90 min. Cells (˜2×10⁶) wereincubated in suspension with control mouse IgG, mouse anti-human α₁β₁(TS2/7), α₂β₁(P1E6), α₃β₁(P1B5), α_(v)β₃(LM609), α₅β₁(P1D6), or β₁(4B4)integrin antibodies, as indicated. Cells then were washed and incubatedwith secondary goat anti-mouse conjugated to FITC (˜5.0 mg/ml), fixed,and subjected to FACS analysis. 96 well microtiter plates were coatedwith increasing concentrations of BSA, FN, and Col I (1.25, 2.5, 5, 10mg/ml), and Scl at 12.5, 25, 50, and 100 nM. Cell adhesion assays wereperformed as described in Example 1. Data are expressed as mean ±SD fromthree replicates and are representative of four independent experiments.

FIGS. 7A–7C demonstrate adhesion and spreading of MRC-5 and WI-38 cellson recombinant Scl protein. Cells detached and suspended in definedmedia were plated on microtiter plates coated with 100 nM of P176 or 1mg/ml of either Col I or FN. After the indicated time points, plateswere washed, fixed and stained. Representative photomicrographs of cellsstained with eosin and hematoxylin are shown following 45 and 90 minutesof adhesion in MRC-5 cells (FIG. 7A) and WI-38 cells (FIG. 7B) at 20×magnification; Bar 200 μm. Cells were grown on coverslips coated withthese substrates (FIG. 7C). After 90 min of permissive adhesion andspreading, cells were fixed and stained with TRITC-phalloidin (i, ii,and iii) and DAPI (iv, v, and vi) to visualize F-actin organization andnucleus. Magnification 100×; Bar 10 μm.

FIGS. 8A–8H show the characterization of and adhesion of fibroblastcells on recombinant P181 (SEQ ID NO: 50) and P182 (SEQ ID NO: 51).Effects of domain swapping between P163 and P176 are demonstrated.Triple-helix formation and two-domain lollipop-like structuralorganization of the recombinant P181 (FIG. 8A) and P182 (FIG. 8D) wereconfirmed by CD spectra (FIGS. 8B and 8E) and EM analyses (FIGS. 8C and8F), respectively. Adhesion activities of MRC-5 and WI-38 cells on wellscoated with either the P176, P181, or P182 substrates was examined.96-well microtiter plates were coated with increasing concentrations ofeither BSA, FN, Col I (1.25, 2.5, 5, 10 □g/ml), or Scl at 12.5, 25, 50,and 100 nM. Adhesion levels of (FIG. 8G) MRC-5 and (FIG. 8H) WI-38 cellsare shown after 45 and 90 min permissive adhesion, as indicated. Dataare expressed as mean ±SD from three replicates and are representativeof four independent experiments.

FIGS. 9A–9C demonstrate that cell adhesion onto immobilized P176 ismediated by α₂β₁ integrin. In FIG. 9A approximately 1×10⁵ MRC-5 cellswere pre-incubated with 1, 5 or 10 μg/ml anti-human [IgG (c), α₁β₁(TS2/7), α₂β₁(P1E6), α₃β₁(P1B5), α_(v)β₃(LM609), α₅β₁(P1D6) and β₁(4B4)]integrin antibodies in PBS containing Ca²⁺/Mg²⁺ on ice for 30 min. Afterwashing with PBS, cells were suspended in defined media and replatedonto microplates pre-coated with 100 nM of P176. Untreated control cells(c) or cells treated with 1, 2 and 5 mM of EDTA were also analyzed.After 45 min, cell adhesion was determined. In FIG. 9B C2C12-α₂+ cellswere plated onto dishes coated with 0.5 μg/ml of Col I, Col IV, VN andFN. Cell adhesion assays were performed after 45 minutes. In FIG. 9CC2C12-α₂+ cells (0.5×10⁶) each were incubated with 10 mg/ml of adhesionblocking monoclonal human anti-integrin antibody for 30 min, asindicated. Cells were washed and resuspended in defined media and werethen replated onto plates coated overnight with 100 nM of Scl P176.Cells not incubated with antibody were included as control (c) or with 5mM EDTA and subjected to cell adhesion for 45 min in a CO₂ incubator.Data are expressed as mean ±SD from four replicates and arerepresentative of two to four independent assays. Values are adjustedagainst background readings obtained from plates incubated in absence ofcells. P-values: *<0.0001; Y<0.001.

FIGS. 10A–10C show surface plasmon resonance (SPR) analyses of α₂integrin subunit I-domain (α₂-I) binding to recombinant Scl proteins.Different concentrations of α₂-I were injected at a flow rate of 20ml/minute for 6 minutes in the presence of 1 mM MgCl₂. Representativesensorgrams of the relative SPR responses of α₂-I over immobilized P176(FIG. 10A) and P181 (FIG. 10B) are shown. The responses of α₂-I over ablank cell were subtracted. Scatchard plot analysis was performed usingthe responses at the steady state portion of the sensorgrams, aspreviously described (65). n_(bound), binding ratio; [P]_(free), freeprotein concentration. The dissociation constants (K_(D)) for α₂-I overP176 (FIG. 10C) in absence of 3 mM EDTA is ˜17 nM. Similar results wereobtained from SPR measurements when P176 was passed over immobilizedα₂-I domain protein (data not shown). Data are representative of thoseobtained from at least two to three separate experiments, with similarresults.

FIGS. 11A–11H demonstrate P176 induced phosphorylation of FAK, paxillin,CAS, and JNK in MRC-5 cells. Serum-starved MRC-5 cells were detachednon-enzymatically, washed, and replated on dishes coated either with FN,Col IV, Col I, or P176. For the phospho-specific immunoblots, i.e.anti-phospho-FAK and anti-phospho-JNK, total cell lysates were analyzed(FIGS. 11A and 11G). To evaluate the tyrosine phosphorylation state ofp130CAS and paxillin, total lysates were pre-adsorbed and subjected toimmunoprecipitation with these antibodies. The resulting immunecomplexes were then subjected to anti-phosphotyrosine immunoblotting(FIGS. 11C and 11E). All samples were then subjected to immunoblottingwith the corresponding non-phospho-specific antibodies, to confirm thatequal amounts of protein were present in each sample (FIGS. 11B, 11D,11F, and 11H). All blots shown are representative of those obtained inat least three separate experiments, with similar results.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment of the present invention there is provided arecombinant triple helical protein comprising a prokaryotic protein orone or more domains of a prokaryotic protein comprising a collagen-likepeptide sequence of repeated Gly-Xaa-Yaa triplets having non-modifiedamino acids. Further to this embodiment the recombinant triple helicalprotein may comprise one or more biologically domains of a mammaliancollagen or peptide therefrom. An example of a mammalian collagen ishuman collagen. In an aspect of this further embodiment, the prokaryoticcollagen-like protein or one or more collagen-like domains thereof arefused to the one or more mammalian collagen domains.

In addition, in this embodiment the recombinant triple helical proteinmay comprise a peptide designed to add or improve a biological functionof the recombinant triple-helical protein without disrupting the triplehelical structure thereof. In one aspect the peptide comprises an aminoacid sequence comprising at least one mutation in one or more of SEQ IDNOS: 1–42 or a peptide fragment thereof. In another aspect the peptidecomprises a peptide fragment thereof or comprising an amino acidsequence generated via computer simulation.

In yet other aspects of this embodiment the collagen-like peptidesequence is from one or more streptococcal collagen-like proteins. Thestreptococcal collagen-like protein(s) may be from a Group AStreptococcus. A representative example of a Group A Streptococcus isStreptococcus pyogenes. Additionally, in this aspect the streptococcalcollagen-like proteins may be a variant of S. pyogenes Scl1 or of S.pyogenes Scl2 or a combination thereof. Further to this aspect thepeptide sequence comprises one or more of SEQ ID NOS: 1–42.

In a related embodiment there is provided a recombinant collagen-likeprotein comprising one or more domains having a peptide sequence of oneor more of SEQ ID NOS: 1–42. Further to this related embodiment therecombinant triple-helical protein further may comprise one or morebiologically active domains of a mammalian collagen. An example is humancollagen. Additionally, in an aspect of this related embodiment, the oneor more of SEQ ID NOS: 1–42 may fused to the one or more human collagendomains. Again further to this embodiment the recombinant collagen-likeprotein may comprise a synthetic peptide as described supra.

In another related embodiment, there is provided a chimericcollagen-like protein comprising one or more domains having a peptidesequence of one or more of SEQ ID NOS: 1–42; and one or morebiologically active domains of a mammalian collagen or peptidetherefrom. The mammalian collagen may be human collagen. Thecollagen-like protein may be a fusion protein or a synthetic protein.Further to this embodiment the chimeric collagen-like protein maycomprise a peptide as described supra.

In another embodiment of the present invention there is provided anexpression vector comprising a DNA sequence encoding the recombinanttriple helical protein described supra. In a related embodiment there isprovided an expression vector comprising a DNA sequence encoding thecollagen-like protein described supra.

In yet another embodiment of the present invention there is provided ahost cell comprising and expressing an expression vector having a DNAsequence encoding the triple helical protein described supra. In arelated embodiment there is provided an expression vector having a DNAsequence encoding the collagen-like protein described supra. In boththese related embodiments the host cell may be a prokaryotic cell.Representative examples of useful prokaryotic cells are well known inthe art and include Escherichia coli, Streptococcus and Bacillus.

In yet another embodiment of the present invention there is provided amethod of producing a triple helical protein comprising introducing intoa prokaryotic host cell an expression vector comprising a DNA sequenceencoding the triple helical protein described supra; culturing theprokaryotic host cell under conditions suitable to express the protein;and isolating the expressed protein thereby producing the triple helicalprotein.

In an aspect of this embodiment the method may further comprisepurifying the triple helical protein. In another aspect the method mayfurther comprise heating the triple helical protein and producing agelatin-like material therefrom. Representative examples of usefulprokaryotic cells are well known in the art and include Escherichiacoli, Streptococcus and Bacillus.

In a related embodiment there is provided a method of producing a triplehelical protein comprising introducing into a prokaryotic cell anexpression vector comprising a DNA sequence encoding a proteincomprising one or more domains having a peptide sequence of one or moreof SEQ ID NOS: 1–42; culturing the prokaryotic host cell underconditions suitable to express the protein; and isolating said expressedprotein thereby producing the triple helical protein. In this embodimentthe method may further comprise the purifying and heating steps asdescribed supra. Representative examples of useful prokaryotic cells arewell known in the art and include Escherichia coli, Streptococcus andBacillus.

In another related embodiment there is provided a method of producing acollagen-like protein comprising introducing into a prokaryotic hostcell an expression vector comprising a DNA sequence encoding thecollagen-like protein comprising one or more domains having a peptidesequence of one or more of SEQ ID NOS: 1–42; culturing the prokaryotichost cell under conditions suitable to express the protein; andisolating the expressed protein thereby producing the collagen-likeprotein. In this embodiment the method may further comprise purifyingand heating steps as described supra. Additionally, the collagen-likeproteins and the prokaryotic host cell are as described supra.

In still another embodiment of the present invention there is provided amethod of producing a gelatin-like material, comprising introducing intoa prokaryotic host cell an expression vector comprising a DNA sequenceencoding a protein comprising one or more domains having a peptidesequence of one or more of SEQ ID NOS: 1–42; culturing the prokaryotichost cell under conditions suitable to express the protein; and heatingthe expressed protein thereby producing the gelatin-like material.Representative examples of useful prokaryotic cells are well known inthe art and include Escherichia coli, Streptococcus and Bacillus.

In still another embodiment of the present invention there is providedan antibody directed against a recombinant streptococcal collagen-like(rScl) protein. In an aspect of this embodiment the rScl protein maybind to α₂β₁ integrin. More specifically, in this aspect the rSclprotein binds to an α₂-I domain of the α₂β₁ integrin. An example of suchan α₂-I domain binding rScl is p176. Thus, in a related aspect of thisembodiment there is provided an antibody directed against p176.Furthermore, in another related aspect the antibody may be directedagainst a domain in p176 that binds an α₂-I domain of α₂β₁ integrin.

In still another embodiment of the present invention there is provided amethod of screening for therapeutic compounds that inhibit aStreptococcal collagen-like protein (Scl) binding to or interacting withan integrin. This method generally comprises measuring Scl binding to orinteraction with the integrin in the presence of a test compound;measuring Scl binding to or interaction with the integrin in the absenceof the test compound in a control sample; comparing binding of theintegrin to Scl in the presence of the test compound with binding in thecontrol sample; and correlating a reduction in binding of the integrinto Scl in the presence of the test compound compared to control withinhibition of integrin binding to or interacting with Scl by the testcompound, thereby screening for the therapeutic compound.

In an aspect of this embodiment the streptococcal collagen-like proteinmay be P176. In another aspect the Scl protein may bind to or interactwith an α₂β₁ integrin. Further to this aspect the Scl protein may bindto or interact with an α₂-I domain of said α₂β₁ integrin.

The following abbreviations are used herein: (r)Scl,(recombinant)streptococcal collagen-like; GAS, group A Streptococcus;FAK, focal adhesion kinase; CAS, crk associated substrate; JNK, c-JunN-terminal kinase; FN, fibronectin; Col I, type I collagen; Col IV, typeIV collagen.

Provided herein are prokaryotic triple helical proteins, prokaryoticcollagen-like proteins and gelatin-like materials comprising theseproteins. Generally, these proteins are recombinant proteins and maycomprise a prokaryotic protein or one or more domains of a prokaryoticprotein comprising a collagen-like peptide sequence of non-modifiedevolutionarily conserved Gly-Xaa-Yaa (GXY) sequence repeats that canform structurally conserved collagen-like triple helices, despite thelack of hydroxyprolines. As such, the GXY triplets may comprise anon-modified proline in the X or Y position.

More particularly, the present invention provides recombinantstreptococcal collagen-like (Scl) proteins, derived from variants ofScl1 and Scl2, i.e., Scl1.41 and Scl2.28 comprising such non-modifiedconserved GXY triplet sequences. One of skill in the art will appreciatethat several sequence characteristics of Scl1 and Scl2 can promote andstabilize the triple helix folding of the CL regions of the twoproteins. For example, the number of continuous GXY repeats is high andoften exceeds the number found in collagenous domains of mammalianproteins. Secondly, 30% and 37% of GXY repeats in Scl1.41 and Scl2.28,respectively, contain prolines, primarily at the X position.

In collagens, the cyclic ring of prolines stabilizes each collagen chainby steric repulsion. Without being bound by theory, the proline residuesin Scls likely have the same effect. Also, Scl proteins have a highcontent of charged amino acids, i.e., 32% and 29.5% in Scl1.41 andScl2.28, respectively, especially lysine and arginine with longionizable side chains that can directly interact with residues of aneighboring polypeptide chain. Further, Scls contain triplets GPR, GER,GPA, GDR, GKD, and GEK, which stabilize a triple helix. None of the GXYtriplets which destabilize the triple helix structure of model syntheticpeptides are present in the CL region of the rScl proteins.

Scls also contain triplets that stabilize the triple helix by formationof alternative hydrogen bonding and hydration patterns. For example, theGEK triplet, which was proposed to stabilize the triple helix throughformation of indirect water-mediated bridges, is the most frequenttriplet in Scl1.41CL. In addition, threonines in the Y position of GETand GKT, found in both Scl1 and Scl2, may substitute for hydroxyprolinesin the formation of hydrogen bonds.

Additionally, the lollipop-like structure of the recombinant Sclproteins resembles the observed structural organization of mammaliancell surface proteins with collagenous domains. Scl proteins likely formlollipop-like structures on the surface of streptococcal cells as do therecombinant polypeptides of the present invention. Scl proteins likelyinteract with molecules in the environment of the streptococci.

The structural organization of the Scl proteins is particularly suitedfor a ligand binding where the stalk-forming CL region projects theglobular V region away from the bacterial surface, facilitating possibleinteractions between the V regions and their potential targets. The CLregions may themselves interact with various molecules as found withmammalian collagens and proteins with collagenous domains. Furthermore,interactions between the CL and V regions may be required in theefficient assembly of the triple helix.

A list of sequences of collagen-like domains from Scl1 and Scl2 proteinsare presented in Table 1. These sequences contain the repeating aminoacid sequence Gly-Xaa-Yaa (GXY) in which glycine occupies every thirdposition which is critical to allow the encoded proteins to form ahomotrimer. The recombinant collagen-like proteins may comprise one ormore domains having a peptide sequence from SEQ ID. NOS: 1–42.

TABLE 1 Strain M Amino Acid Sequence in the collagen- No type likeregion of Scl1 protein 6708 M1 GKSGIKGDRGETGPAGPAGPQGKTGERGAQGP (SEQ IDNO: 1) KGDRGEQGIQGKAGEKGERGEKGDKGETGERGEKGEAGIQGPQGEAGKDGAPGKDGAPGEKGEKGD RGETGAQGPVGPQGEKGETGAQGPAGPQGEAGKPGEQGPAGPQGEAGQPGEK 3803 M2 GEKGEAGIQGPQGKAGKDGAPGKDGAVGAQGP (SEQ ID NO:2) KGDKGDTGEKGETGATGAQGPQGEAGKDGAPG EKGEKGDRGETGAQGPVGPQGEKGETGAQGPAGPQGEKGETGAQGPAGPQGEAGKPGEQGPAGP QGEAGKPGEK 315 M3GDKGETGLAGPVGPAGKAGARGAQGPAGPRG (SEQ ID NO: 3) 321 M4GEKGDAGPRGERGPQGPVGPAGKAGEKGEAGI (SEQ ID NO: 4)QGPQGEAGKDGAPGKDGAPGEKGEKGDRGETG AQGPVGPQGEKGETGAQGPAGPQGEAGKPGEQGPAGPQGEAGKPGEQGGKPGEK 6169 M6 GEKGDPGAQGPKGEKGEKGDRGDTGAQGPVGP (SEQ IDNO: 5) QGEAGQPGEK 6159 M9 GEKGDAGPVGPAGPRGERGPQGEKGAQGLKGE (SEQ ID NO:6) KGDTGAVGAQGPKGDKGDTGERGEKGDTGATG AQGPQGEAGKDGAPGKDGAPGEKGEKGDRGETGAQGPVGQQGEAGKPGEQGEAGKPGEQ 6276 M12 GPAGPKGETGPAGPAGPEGKPGKAGEKGDRGE(SEQ ID NO: 7) KGEAGIQGPQGEKGDTGAQGPQGEAGAPGEKGEKGDRGETGAQGPVGPQGEKGETGAQGPQGEA GKPGEQGPQGEAGKPGEK 6259 M12GPAGPKGETGPAGPAGPEGKPGKAGEKGDRGE (SEQ ID NO: 8)KGEAGIQGPQGEKGDTGAQGPQGEAGAPGEKG EKGDRGETGAQGPVGPQGEKGETGAQGPQGEA GKPGEK6144 M12 GPAGPKGETGPAGPAGPEGKPGKAGEKGDRGE (SEQ ID NO: 9)KGEAGIQGPQGEKGDTGAQGPQGEAGKPGEKA PEKSPEGEAGQPGEK 156 M18GKSGIKGDRGETGPAGPAGPQGKTGERGAQGP (SEQ ID NO: 10)KGDRGEQGIQGKAGEKGERGEKGDKGETGERG EKGEAGIQGPQGEAGKDGAPGKDGAPGEKGEKGDRGETGAQGPVGPQGEKGETGAQGPAGPQGE AGKPGEQGPAGPQGEAGQPGEK 6269 M22GPAGPEGKPGPKGDKGETGARGPRGERGETGL (SEQ ID NO: 11)QGPKGEAGKDGAQGEKGEKGDRGEKGEAGIQG PKGEAGKDGAPGEKGEKGDRGETGAQGPVGPQGEKGETGAQGPAGPQGEAGKPGEQGPAGPQGE AGKPGEK 6141 M28GDKGDAGPKGERGPAGPQGPVGPKGEAGKVGA (SEQ ID NO: 12)QGPKGDPGAPGKDGAKGEKGDKGDTGERGEKG DIGATGAQGPAGPQGEAGKPGEQGPAGPQGEAGKPGEKAPEKSPEGEAGQPGEK 6143 M28 GDKGDAGPKGERGPAGPQGPVGPKGEAGKVGA (SEQ IDNO: 13) QGPKGDPGAPGKDGAKGEKGDKGDTGERGEKGDIGATGAQGPQGEAGKDGAPGEKGDKGDRGET GAQGPVGPQGEKGETGAQGPAGPQGEAGKPGEQGPAGPQGEAGKPGEK 6274 M28 GDKGDAGPKGERGPAGPQGPVGPKGEAGKVGA (SEQ ID NO:14) QGPKGDPGAPGKDGAKGEKGDKGDTGERGEKG DIGATGAQGPQGEKGETGAQGPAGPQGEAGKPGEQGPAGPQGEAGKPGEK 6183 M41 GEKGEAGPQGEKGLPGLTGLPGLPGERGPRGP (SEQ ID NO:15) KGDRGETGAQGPVGPQGEKGEAGTPGKDGLRG PQGDPGAPGKDGAPGEKGDRGETGAQGPVGPQGEKGEAGTPGKDGAPGEKGEKGDRGETGATGA QGPQGEAGKDGAQGPVGPQGEKGETGAQGPAGPQGEKGETGAQGPAGPQGEAGQPGEK 4569 M49 GPKGDPGPVGPRGPEGKPGKDGAKGDTGPRGE(SEQ ID NO: 16) RGEQGIQGEQGKAGEKGEKGDKGDTGERGEKGDTGATGAQGPQGEAGKDGAPGEKGEKGDTGAQ GPVGPQGEKGETGAQGPQGEAGKPGEQGPAGPQGEAGKPGEK 6186 M52 GPKGDPGPAGPRGPVGPEGPAGKPGKDGAQGE (SEQ ID NO: 17)RGKQGDPGPKGDKGEDGKVGPRGPKGDRGETG AQGPVGPQGETGKDGAPGEKGEKGDRGETGAQGPVGPQGETGKDGAPGEKGEKGDRGETGAQGP VGPQGETGKDGAPGEKGEKGDRGETGAQGPVGPQGEKGETGAQGPAGEK 6177 M52 GPKGDPGPAGPRGPVGPVGPVGPAGKPGKDGA (SEQ ID NO:18) QGERGKQGDPGPKGDKGEDGKVGPRGPKGDRG ETGAQGPVGPQGETGKDGAPGEKGEKGDRGETGAQGPVGPQGETGKDGAPGEKGEKGDRGETGA QGPVGPQGETGKDGAPGEKGEKGDRGETGAQGPVGPQGEKGETGAQGPAGEK 1863 M55 GEKGDPGAPGKDGAVGAQGPKGEKGEKGDRGD (SEQ IDNO: 19) TGAQGPVGPQGEKGEKGEKGEKGETGEQGPAG PQGEAGKPGEK 4487 M56GIKGDRGETGPAGPAGPVGPVGPRGPEGPEGK (SEQ ID NO: 20)PGKRGAQGIQGPKGDKGETGERGEQGLQGEKG DTGAAGAPGKDGVQGPKGDKGETGERGEKGEAGIQGPQGEKGDTGATGAQGPQGEAGKDGAPGE KGEKGDRGETGAQGPVGPQGEKGETGAQGPAG EK6146 M56 GIKGDRGETGPAGPAGPVGPVGPRGPEGPEGK (SEQ ID NO: 21)QGKPGKRGAQGIQGPKGDKGETGERGEQGLQG EKGDTGAAGAPGKDGVQGPKGDKGETGAQGPVGPQGEKGETGAQGPAGEK 1864 M57 GDKGDAGPKGERGPAGPQGPVGPKGEAGKPGA (SEQ ID NO:22) QGPKGDKGETGERGETGAQGPVGPQGEKGETG EQGPAGPQGEAGKPGEQGPAGQPGEK 4673 M75GDKGDTGPAGPQGKTGERGAQGPKGDRGEQGI (SEQ ID NO: 23)QGKAGEKGERGEKGDKGETGERGEKGEAGIQG PQGEKGDTGAQGPQGEAGKDGAPGEKGEKGDRGDTGAQGPVGPQGEKGETGAQGPAGPQGEAGQ PGEK 6133 M76GKSGIKGDRGETGPAGPAGPRGPVGPAGEAGK (SEQ ID NO: 24)QGDRGEQGIQGPKGEAGAPGKDGAKGEKGDKG DTGERGEKGDTGAQGPQGEAGKDGAPGKDGAPGEKGEKGDRGETGAQGPVGPQGEKGETGAQGP AGPQGEAGKPGEQGPAGPQGEAGKPGEK 6191 M77GPAGPAGPRGPKGEDGKAGAPGKDGAPGKDGA (SEQ ID NO: 25)PGKDGAPGKDGAQGPKGDKGETGERGEKGETG ATGAQGPQGEAGKDGAPGEKGEKGDRGETGAQGPVGPQGEKGETGAQGPAGPQGEAGQPGEQGP AGPQGEAGQPGEK 6250 M77GPAGPAGPRGPKGEDGKAGAPGKDGAPGKDGA (SEQ ID NO: 26)PGKDGAQGPKGDKGETGERGEKGETGATGAQG PQGEAGKDGAPGKDGAPGKDGAQGPKGDKGETGERGEKGETGATGAQGPQGEAGKDGAPGEKGE KGETGAQGPAGPQGEAGQPGEQGPAGPQGEAG QPGEK1880 ST2035 GEKGEAGIQGPQGKAGKDGAPGKDGAPGKDGA (SEQ ID NO: 27)VGAQGPKGDKGDTGEKGETGATGAQGPQGEAG KDGAPGEKGEKGDRGETGAQGPVGPQGEKGETGAQGPAGPQGEAGKPGEQGPAGPQGEAGKPGE K 6155 ST2967GKSGIKGDRGEAGPAGPAGPRGERGPAGEAGK (SEQ ID NO: 28)QGERGEQGIQGPKGETGAVGAQGPKGDKGDTG ERGEKGDTGATGAQGPQGEAGKDGAPGEKGEKGDRGETGLQGPVGPQGEKGEIGAQGPAGPQGE AGIPGEK Strain M Amino Acid Sequence inthe collagen- No type like region of Scl2 protein 6708 M1GPKGPAGEKGEQGPTGKQGERGETGPAGPRGD (SEQ ID NO: 29)KGETGDKGAQGPVGPAGKDGQDGKDGLPGKDG KDGQDGKDGLPGKDGK 3803 M2GDQGERGEAGPQGPAGQDGKAGDRGETGPAGP (SEQ ID NO: 30)VGPAGPQGPRGDKGETGERGEQGPAGQDGKAG DRGETGPAGPVGPAGPQGPRGDKGETGERGEQGPAGQDGKDGDRGETGPAGPVGPAGKDGQDGK DGLPGKDGKDGQDGKDGLPGKDGKDGQPGKP 315 M3GQDGDRGEAGPAGPRGEAGPAGPRGEAGKDGA (SEQ ID NO: 31)KGDRGEAGPAGPRGEAGKDGAKGDRGEAGPAG PRGEAGKDGAKGDRGEAGPAGPRGEAGKDGAKGDRGEAGPAGPRGEAGPAGPRGEAGPAGPRGE AGPAGPRGEAGKDGAKGDRGEAGPAGPRGEAGKDGAKGDRGEAGPAGPRGEAGKDGAKGDRGEA GPAGPRGEAGPAGPRGEAGKDGAKGDRGEAGPAGPRGEAGKDGAKGDRGEAGPAGPRGEAGKDG AKGDRGEAGPAGPRGEAGKDGAKGDRGEAGPAGPRGEAGKDGAKGDRGEAGPAGPRGEAGKDGA KGDRGEAGPAGPRGEAGPAGKDGQPGKP 321 M4GDKGEPGAQGPAGPRGETGPAGERGEKGEPGT (SEQ ID NO: 32)QGAKGDRGETGPAGPRGDKGEKGEQGPAGKDG ERGPIGPAGKDGQDGKDGLPGKDGKDGQDGKDGLPGKDGKDGQDGKDGLPGKP 6159 M9 GDQGDPGERGETGPAGPAGPVGPVGPRGERGE (SEQ IDNO: 33) AGPAGQDGKAGDRGETGPAGPVGPRGDKGEKGEQGPAGKDGLPGKDGKDGQDGKDGLPGKDGKD GQDGKDGLPGKDGKDGQDGKDGLPGKDGKDGQDGKDGLPGKDGKDGQDGKDGLPGKDGKDGQDG KDGLPGKDGQPGKP 6139 M12GEKGERGPVGPAGPQGLQGTKGDRGETGEQGQ (SEQ ID NO: 34)RGETGPAGPQGPAGPVGPAGKDGEAGAQGPVG PAGKDGQDGKDGLPGKDGQDGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQDGKDGQ DGKDGLPGKDGQDGKDGLPGKDGQDGKDGKDGLPGKDGKDGLPGKDGKDGQPGKP 6276 M12 GEKGERGPVGPAGPQGLQGTKGDRGETGEQGQ (SEQID NO: 35) RGETGPAGPQGPAGPVGPAGKDGQDGKDGLPGKDGQDGKDGLPGKDGQDGKDGLPGKDGKDGQD GKDGLPGKDGKDGLPGKDGPDGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQDGKDG QDGKDGLPGKDGQDGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQPGKP 6143 M28 GQDGRDGERGEQGPTGPTGPAGPRGLQGLQGE (SEQID NO: 36) RGEQGPTGPAGPRGLQGERGEQGPTGLAGKAGEAGAKGETGPAGPQGPRGEQGPQGLPGKDGEA GAQGPAGPMGPAGERGEKGEPGTQGAKGDRGETGPVGPRGERGEAGPAGKDGERGPVGPAGKDG QDGQDGLPGKDGKDGQDGKDGLPGKDGKDGQDGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGK DGKDGQPGKP 6274 M28GQDGRDGERGEQGPTGPTGPAGPRGLQGLQGL (SEQ ID NO: 37)QGERGEQGPTGPAGPRGLQGERGEQGPTGLAG KAGEAGAKGETGPAGPQGPRGEQGPQGLPGKDGEAGAQGPAGPMGPAGERGEKGEPGTQGAKGD RGETGPVGPRGERGEAGPAGKDGERGPVGPAGKDGQDGQDGLPGKDGKDGQDGKDGLPGKDGKD GQDGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQPGKP 6146 M56 GKDGETGPAGPTGPAGAKGETGPAGPVGPRGD (SEQ ID NO: 38)KGEKGEQGPAGKDGLPGKDGKDGQDGKDGLPG KDGKDGQDGKDGLPGKDGKDGQDGKDGLPGKDGKDGKDGKDGQPGKP 4487 M56 GKDGETGPAGPTGPAGAKGETGPAGPVGPRGD (SEQ ID NO:39) KGEKGEQGPAGKDGLPGKDGKDGQDGKDGLPG KDGKDGQDGKDGLPGKDGKDGQDGKDGLPGKDGKDGKDGKDGQPGKP 4673 M75 GDQGEPGEPGEPGERGPRGEVGPAGPQGPVGP (SEQ ID NO:40) VGPAGKDGTQGPRGDKGEPGEQGQRGETGPAG PQGPAGPVGPAGKDGTQGPRGDKGETGEQGQRGEVGPAGPQGPVGPVGPAGKDGAKGDRGETGP AGPAGKDGEAGAQGPGPAGPQGPRGDKGETGDKGEQGPAGKDGERGPVGPAGKDGQDGLPGKDG KDGQDGKDGLPGKDGKDGQDGKDGLPGKP 6191 M77GPRGDKGETGEQGPRGAQGPAGPQGPVGPAGK (SEQ ID NO: 41)DGTQGPRGDKGETGEQGPRGAQGPAGPQGPVG PAGKDGTQGPRGDKGETGEQGPRGAQGPAGPQGPMGPAGERGEKGEPGTQGAKGDRGETGPAGP VGPRGDKGETGAKGEQGPAGKDGKAGERGPVGPAGKDGQDGKDGLPGKDGKDGQDGKDGLPGKD GKDGQDGKDGLPGKDGKDGQDGKDGLPGKDGKDGKDGKDGQPGKP 6155 St2967 GAKGEAGPAGPKGPAGEKGERGETGPAGPAGK (SEQ ID NO:42) DGEAGAQGPMGPAGPQGPRGDKGETGDKGDQG PAGKDGDRGPVGPQGPQGETGPAGPAGKDGEKGEPGPRGEAGAQGPAGPQGPRGDKGETGDKGE QGPAGKDGERGPVGPAGKDGQDGKDGLPGKDGKDGQDGKDGLPGKDGKDGQDGKDGLPGKDGKS GQPGKP

The present invention also provides chimeric triple helical proteins orchimeric collagen-like protein. The recombinant triple helical proteinsor recombinant collagen-like proteins described herein may additionallycomprise at least one biologically active domain from a mammaliancollagen, such as, but not limited to human collagens. For example, theprotein may comprise one or more of SEQ ID NOS.: 1–42 and one or moredomains from a mammalian, e.g., human, collagen protein.

The chimeric protein may be a fusion protein. Methods of constructingvectors suitable to transfect and express DNA encoding a fusion proteinare standard in the art and well known to those of ordinary skill in theart. Alternatively, the protein may be a synthetic protein.

It is further contemplated that a peptide may be added to the triplehelical recombinant proteins, the recombinant collagen-like proteins orthe chimeric collagen-like proteins described herein. Addition of thesepeptides may alter structure and thereby function of the chimericprotein in a specifically selected manner. For example a new functionmay be added or an existing function improved. However the triplehelical structure inherent in all the proteins described herein is notdisrupted by the presence of the this peptide. The peptides may compriseat least one amino acid mutation in at least one of SEQ ID NOS.: 1–42 orin a peptide fragment thereof. Alternatively, the sequence of thepeptide may be determined via computer simulation.

As such, the present invention further provides methods of producing atriple helical protein or a collagen-like protein. The methods mayutilize expression vectors comprising a DNA that encodes the triplehelical or collagen-like proteins of the present invention.Additionally, the present invention provides a host cell, such as aprokaryotic cell, for example, although not limited to, Escherichiacoli, a Streptococcus or a Bacillus. It is standard in the art toconstruct such expression vectors and to transform a host cell toexpress the product of the DNA contained therein.

Generally, an expression vector as described herein is introduced intothe prokaryotic host cell. The prokaryotic cell is cultured underconditions suitable to induce expression of the protein. The expressedprotein is isolated and may further be purified. Also provided is amethod of producing a gelatin-like material from the recombinantproteins presented herein. The triple helical and/or the collagen-likeproteins may be heated to produce the gelatin-like material.

Furthermore, the present invention contemplates that designer collagensor gelatin-like substances may be produced for the specific needs of theuser using the recombinant triple helical proteins, collagen-likeproteins or chimeric proteins described herein. For example a collagenor gelatin may be designed to effectively function in any biologicalsystem. The problems associated with mammalian collagens, such as bovinecollagens, can be avoided. This effectively would change and improve thecommercial use of collagens.

The present invention further demonstrates that a member of theprokaryotic collagen-like proteins, the Scl1.41 variant of S. pyogenes,interacts with the I-domain of α₂β₁ integrin, but not α₁β₁ integrin.This interaction induces cell adhesion and signaling activities throughFAK and CAS in human lung fibroblast MRC-5 cells. Thus, the Scl1.41recombinant protein P176 (SEQ ID NO.: 44) can interact with an integrinin a productive manner, preferably via a binding site in the CL domainshown in SEQ ID NO.: 15.

Furthermore it is contemplated that cell adhesion to P176 ispredominantly mediated by integrins. First, removal of Ca²⁺ and Mg²⁺metal ions, which are required for collagen-integrin binding, inhibitedcell adhesion activity. Second, the addition of an anti-β₁ integrinantibody completely inhibited adhesion of these cells onto P176. Third,cell adhesion to P176 was specifically mediated by the α₂β₁ integrin, aknown type I collagen receptor, because an anti-α₂β₁ integrin monoclonalantibody blocked cell adhesion, whereas an anti-α₁β₁ antibody did not.Fourth, the specificity of the α₂β₁-P176 interaction was furthersupported by the fact that C2C12-α₂+ cells adhered to P176 and ananti-α₂β₁ integrin antibody or EDTA inhibited this adhesion. Finally,SPR analysis showed that P176 interacts with the I-domain of α₂integrin, which is an important characteristic of a collagen-integrinbinding (34).

However, integrin ligation is not a general property of Scl proteins andseveral other recombinant proteins based on different Scl sequences didnot support cell adhesion, such as the Scl2.28 recombinant protein P163,for example. This suggests that specific sequences present in some butnot other Scl proteins are recognized by the integrins. It has beendemonstrated that the α₁β₁, and α₂β₁ integrins interact with specificsequences in mammalian collagens. The substrate specificity of the twointegrins appears to be similar, but not identical. Thus, α₁β₁ but notα₂β₁, integrin requires a hydroxyproline residue in the binding site forfull activity. Consistent with this observation α₂β₁, but not α₁β₁,mediates cell adhesion to the P176 protein that does not containhydroxy-proline residues.

Given that integrins are involved in streptococci host cell invasion andthat fibronectin binding a MSCRAMM peptide (SEQ ID NO.: 46) ongram-positive bacteria can recruit soluble fibronectin which is thenrecognized by α₅β₁ integrin thereby initiating cellular invasion, itcould be contemplated that integrin interaction through a Scl proteinwould play a role in the infections process of S. pyogenes.Additionally, the α₂β₁ integrin is also a prominent collagen receptor onplatelets and it is possible that Scl proteins may induce a plateletsignaling through interacting with the platelet integrin. The Sclprotein-integrin interaction described herein suggest that thesebacterial proteins are mimicking and being recognized as collagens whenit comes to α₂β₁ integrin. It is further contemplated that Scl proteinscould also behave as collagens in other systems and that this molecularmimicry allows the bacteria to manipulate host biology at a number ofdifferent levels.

Thus the present invention provides antibodies directed againstintegrin-binding or integrin-interacting rScl proteins described herein.Preferably, cell adhesion is mediated by α₂β₁ integrin. Most preferably,the rScl proteins bind to the I-domains of the integrins, such as to theα₂-I domain of α₂β₁ integrin. The antibody may be directed against P176or to the domain in P176 that binds α₂β₁ integrin or, more specifically,binds the α₂-I domain of α₂β₁ integrin. It is contemplated thatantibodies may be directed against rScls comprising a P176 α₂-I domainbinding site or a P176-like CL domain or P176-like α₂-I domain bindingsite.

The present invention further provides means to screen for therapeuticcompounds or drugs, including proteins or peptides, that inhibit P176from binding or interacting with integrin I-domains. Generally, apotential therapeutic compound may be screened by comparing the bindingof α₂-I to P176 in the presence and absence, as control, of thepotential therapeutic compound. Inhibition of α₂-I binding in thepresence of the test compound would screen for potential therapeuticefficacy.

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion.

EXAMPLE 1 Materials and Methods

Recombinant Proteins

Recombinant proteins were obtained using the Strep-tag II expression andpurification system (IBA-GmbH). Fragments encoding sequences of thescl1.1 allele were amplified from the serotype M1 strain MGAS6708identical to SF370 used for genome sequencing. The scl2.28 sequence wasamplified from the serotype M28 strain MGAS6274. DNA fragments wereligated to the Escherichia coli vector pASK-IBA2, and clones wereconfirmed by sequencing. Recombinant proteins were expressed in E. coliand purified by affinity chromatography on a Strep-Tactin-Sepharose.Thus, pSL163 was constructed which encodes a P163 polypeptide. Theidentity of each purified protein was confirmed by amino-terminalsequencing, and mass determinations were done by electrospray ionizationmass spectrometry measurements.

Western immunoblot analysis was performed with proteins separated onSDS-PAGE, transferred onto a nitrocellulose membrane (AmershamBiosciences) and probed with specific, affinity-purified polyclonalrabbit antibodies raised against a synthetic peptide derived from the Vregion of Scl1.1. Horseradish peroxidase-conjugated goat anti-rabbitaffinity-purified immunoglobulin G, heavy and light chains, (Bio-Rad)was used as the secondary antibody, and SuperSignal chemiluminescentsubstrate (Pierce) was used in signal detection. Prestained broad-rangemarker proteins (Bio-Rad) were used as molecular mass standards.

To obtain recombinant P176, the DNA sequence of the scl1.M41 allele(accession number AY452037) was amplified and cloned into an E. colivector pASK-IBA2, resulting in plasmid pSL176. In a domain swappingexperiment, plasmid pSL163 was used as a core sequence. First, a DNAfragment encoding the variable V region of P176 was amplified andsubstituted for the corresponding region of pSL163, resulting in thepSL181/P181 construct. Next, the pSL182 plasmid was obtained byreplacing the DNA sequence of the collagen-like region in pSL181,originally from pSL163, with a corresponding DNA sequence of pSL176,which lacked a linker region. These plasmid constructs were verified byDNA sequencing. As above, recombinant proteins were purified by affinitychromatography on Strep-Tactin-Sepharose and their identity wasconfirmed by amino-terminal sequencing.

Gel Migration Analyses

The ability of the Scl proteins to form oligomers was examined by gelelectrophoresis. Protein samples were heat-denatured at 90° C. for 5 minand then placed on ice for 2 min before loading, whereas native proteinsamples were not heated and were kept at 4° C. Denatured or native P144and P163 proteins were electrophoretically separated in 10–20% gradientpolyacrylamide gels with or without 0.1% SDS and visualized by staining.

The resistance of triple helix collagen to trypsin digestion was used asan indicator of macromolecular structure. Native and heat-denaturedsamples of recombinant P144 dissolved in 25 mM HEPES, pH 8.0, weredigested with 10 μg/ml trypsin using an enzyme:protein molar ratio of1:10 at 15° C. Aliquots were removed after 15, 30, and 60 min, and thereaction was stopped with phenylmethylsulfonyl fluoride. Samples wereanalyzed by SDS-PAGE using undigested P144 and P157 as size markers.Resistance of the triple-helical domain to trypsin was measured fromband intensities and compared with an undigested sample.

Circular Dichroism (CD) Spectroscopy

The triple helical conformation of recombinant rScls was analyzed bycircular dichroism, as described (62). Spectra were recorded on a JascoJ720 spectropolarimeter equipped with a variable temperature unit.Samples were dissolved in 1% phosphate-buffered saline, pH 7.4. Thermaltransition profiles were recorded at 220 nm, as described above, with atemperature slope of 20° C./h or of 10° C./h. Measurements were takenwith a 0.5-cm path length and data were integrated for 1 sec at 0.2-nmintervals with a bandwidth of 1 nm. A wavelength scan was performed foreach protein before unfolding (4° C., 25° C.) and after unfolding (50°C.), or after subsequent refolding (4° C., renatured). The meltingtemperatures (t_(m)) were given as mean ±S.D. of t_(m) values fromseveral measurements. Secondary structure compositions were estimatedusing three deconvolution programs, CD Estima, CONTIN, and SELCON, andthe results were averaged.

Electron Microscopy

The structural organization of the recombinant proteins was viewed byelectron microscopy of the rotary shadowed rScls, as previously (63).Protein samples (100 μg/ml) were dialyzed against 0.1 M ammoniumbicarbonate and then mixed with glycerol to a final glycerolconcentration of 70% v:v. Samples were sprayed onto freshly cleaved micasheets and rotary shadowed with carbon/platinum at an angle of 6degrees. The replicas were backed with carbon at 90 degrees and placedon copper grids. Photomicrographs were taken with a Philips 410 electronmicroscope operated at 80 KV with a 30-μm objective aperture.

The microscope was calibrated using a carbon grating replica. The heightof the goniometer stage was adjusted for each grid square before takingpictures and the lenses were saturated before taking each photo.Molecule contours were traced on a digitizing tablet at a finalmagnification of ×181,500 and measurements were generated with Bioquantsoftware.

Reagents and Antibodies

Cell culture media, human fibronectin, type I and type IV collagen,control mouse IgG, and anti-α₁β₁ (TS2/7), anti-α₂β₁ (P1E6), anti-α₃β₁(PiB5), anti-α₅β₁ (P1D6) integrin antibodies were purchased fromGibco-BRL/Invitrogen (Carlsbad, Calif.). Anti-FAK (2A7), anti-paxillinand anti-phospho-tyrosine (4G10) mouse monoclonal antibodies (mAbs) werepurchased from Upstate Biotechnology Inc. (Lake Placid, N.Y.).Anti-phospho-JNK and anti-phospho-FAK (Y397) antibodies were purchasedfrom Cell Signaling Technology (Beverly, Mass.). The anti-α_(v)β₃(LM609) was obtained from Chemicon (Temecula, Calif.). Anti-β₁ (4B4)integrin antibody was procured from BD Biosciences (San Jose, Calif.).

Cells and Cell Culture

Human lung fibroblast cell lines MRC-5 and WI-38 were obtained from ATCC(CCl-171; CCL-75) and cultured in DMEM supplemented with 10% FBS, 2.4 mML-glutamine and antibiotics. Parental mouse myoblast C2C12 (controlcells) and C2C12 cells stably transfected with the human α₂-integrinsubunit (C2C12-α₂+), provided by D. Gullberg (Uppsala, Sweden), weremaintained in 10% FBS in DMEM in the absence or presence of 10 mg/mlpuromycin, respectively. Methods for growth and maintenance of adherentcells have been described (56, 64).

Fluorescent Activated Cell Sorting (FACS)

Cells were washed in PBS, pH 7.4, then non-enzymatically dissociatedwith 2 mM EDTA in PBS and washed twice in PBS containing Ca²⁺ and Mg²⁺.Cells were passed through a cell strainer and enumerated. 2×10⁶ cellswere then incubated with saturating amounts of control mouse IgG oranti-human integrin monoclonal antibodies, e.g., anti-α₁β₁ (TS2/7),anti-α₂β₁ (P1E6), anti-α₃β₁ (P1B5), anti-α_(v)β₃ (LM609), anti-α₅β₁(P1D6) and anti-β₁ (4B4) for 30 minutes at 4° C. Secondary labeling wasperformed for 1 hour at 4° C. with a goat-anti mouse IgG conjugated tofluorescein isothiocyanate (FITC) (Molecular Probes, OR). Cells werewashed twice with PBS, fixed with 0.5% p-formaldehyde (PFA) and analyzedwith the FACS Calibur flow cytometer (Becton Dickinson, San Jose,Calif.).

Cell Adhesion and Spreading Assays

Cell adhesion to 24-well plates coated with specified substrates hasbeen described (64). Briefly, the test substrates, i.e., Scl recombinantproteins, were prepared at a concentration of 12.5, 25, 50 and 100 nM inTBS (20 mM Tris, pH. 7.4, 150 mM NaCl, 0.02% sodium azide) and coatedonto sterile microplates overnight at 4° C. Human fibronectin, type IVcollagen, type I collagen and BSA (1.25, 2.5, 5 and 10 μg/ml) wereincluded as controls. BSA was subjected to 0.22 μm filtration and heatinactivation. After washing with PBS, plates were saturated with 0.5%BSA for 60 min in a 37° C. CO₂ incubator.

Human lung fibroblast MRC-5 and WI-38 or C2C12 cells starved overnightin serum-free media were detached in PBS containing 2 mM EDTA and0.0025% trypsin. Cells were collected and resuspended in M199 definedmedia containing appropriate antibiotics, 0.2% BSA and 1×ITS (insulin,transferrin and selenium-A), supplemented with 2 mM Mg²⁺ and 1 mM eachof Ca²⁺/Mn²⁺. Saturated plates were washed once with PBS and 100 μl ofcell suspension (0.15×10⁶ cells/ml) were added and allowed to adhere.

After 45 or 90 min, unattached cells were gently removed and washed withPBS before fixing in 3% PFA for 10 min. The cells were then washed withcold TBS, refixed in 20% methanol for 10 min, and stained in 0.5%crystal violet for 5 min. Plates were thoroughly washed with water andair-dried. The dye was then eluted in 100 μl of 100 mM sodium citrateand absorbance was measured at 590 nm.

To visualize cell spreading, PFA-fixed cells were stained with standardeosin and hematoxylin and pictures were captured in an inverted lightmicroscope under 20× magnification. Alternatively, cells grown oncoverslips under similar conditions were stained with phalloidin-TRITC(Sigma, P-1951) and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI;Molecular Probes, D-1306) to identify stress fibers and nuclei,respectively. A Zeiss Axiovert-125 fluoroscope was used for thispurpose.

Cell Adhesion Blocking Assay

The cell adhesion blocking assay has been described (64). In brief, 70%confluent human lung fibroblast (MRC-5), parental C2C12 and C2C12-₂+cells were washed in PBS, detached non-enzymatically in 2 mM EDTA, andwashed. Cells were passed through a cell strainer and enumerated.Approximately 0.1×10⁶ cells were incubated with 1, 5 and 10 mg/ml ofanti-a₁b₁, anti-a₂b₁, anti-a₃b₁, anti-a₅b₁, anti-a_(v)b₃ and anti-b₁integrin monoclonal antibodies on ice for 30 min. Cells were washed,resuspended in defined media, and seeded onto dishes pre-coated with 100nM P176 and subjected to cell adhesion assays. The use of single dose(10 mg/ml) anti-integrin monoclonal antibody for C2C12-a₂+ cell adhesionblocking assay was considered optimal. Cold (4° C.) PBS, pH 7.4containing 1 mM of Ca²⁺ and Mg²⁺ was used for washing cells. Statisticalanalyses were performed as described (64).

Recombinant I-Domain of α₂ Integrin Subunit

Cloning, expression, and purification of the I-domain of α₂ integrinhave been described previously (65). In brief, a DNA fragment encodingthe I-domain of α₂ integrin subunit was amplified by PCR from a humanhepatoma cDNA library and subcloned into the expression vector pQE30(Qiagen Inc., Chatsworth, Calif.). The accuracy of the DNA sequence wasverified by dideoxy DNA sequencing (Lonestar Lab). Large-scaleexpression and purification of the recombinant α₂-I were carried outusing Ni⁺²-chelating affinity chromatography, as described (62).

Surface Plasmon Resonance (SPR) Analysis

Measurements were performed at room temperature in a BIACORE 3000instrument (BIAcore, Uppsala, Sweden) as described previously (65).Briefly, 990 response units (RU) of P176Scl and 717 RU of P181 Sclproteins were immobilized onto the flow cells on a CM5 chip. Differentconcentrations of α₂-I proteins in HBS (25 mM HEPES, 150 mM NaCl, pH7.4) buffer containing 5 mM β-mercaptoethanol, 1 mM MgCl₂ and 0.05%octyl-D-glucopyranoside were passed over these surfaces at 20 μl/min for6 min. Regeneration of the Scl protein surface was achieved by running10 μl of a solution of 0.01% SDS. Binding of α₂-I to a reference flowcell, which had been activated and deactivated without the coupling ofproteins, was also measured and was subtracted from the binding to Sclprotein-coated chips.

SPR sensorgrams from different injections were overlaid using theBIAevaluation software (BIAcore AB). Data from the equilibrium portionof these sensorgrams were used for Scatchard analysis. Based on thecorrelation between the SPR response and change in soluble I-domainprotein binding to the immobilized Scl proteins, values for the bindingratio, n_(bound), and the concentration of free protein, [P]free, werecalculated using the equations described previously (65). Scatchardanalysis was performed by plotting n_(bound)/[P]_(free) againstn_(bound), in which the negative reciprocal of the slope is thedissociation constant, K_(D) and the X-intercept is the number ofbinding interactions, n.

Biochemical Methods

Cells were serum-starved overnight, detached and maintained insuspension (Sus) for 45 minutes at room temperature. Cells were thenreplated onto dishes coated with indicated substrates. After 30 or 60min cells were washed with cold PBS, pH 7.4, and solubilized in cellextraction buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton X-100, 10mM sodium fluoride, 1 mM sodium pyrophosphate, 2 mM sodium orthovanadateand with various protease inhibitors freshly added) for 30 minutes onice. Extracts were centrifuged at 21,000×g for 30 minutes at 4° C. toremove insoluble material.

Protein concentrations of the resulting lysates were determined by theBio-Rad DC protein assay. For immunoprecipitation analyses, cell lysateswere pre-adsorbed with mouse IgG-agarose beads at 4° C. for 1 hour.Immunoprecipitation, immunoblotting, and detection protocols wereperformed as described previously (64, 66). For immunoprecipitation, 2–3mg of antibodies were used for each sample. For immunoblotting analyses,antibodies were prepared at a concentration of 0.5–2 mg/ml in 1×TBS, pH7.4, with 3% BSA.

EXAMPLE 2

Characterization of Recombinant Scl Proteins

Previous studies demonstrated that recombinant proteins expressed ineukaryotic systems could successfully be used to study the structure andorganization of mammalian proteins with collagenous domains. A series ofrecombinant polypeptides generated in a prokaryotic (E. coli) expressionsystem derived from either the Scl1.1, i.e., a Scl1 protein from aserotype M1 GAS, or the Scl2.28, i.e., a Scl2 protein from a M28-typeGAS, protein that contain collagen-like regions composed of continuousGXY repeats was examined (FIG. 1A).

These two related proteins were selected for structural studies becausethey were both expressed by the parental GAS isolates and they differedsignificantly in their primary amino acid sequence. The collagen-likeregion (CL) is composed of 50 and 79 GXY repeats in Scl1.1 and Scl2.28,respectively. The amino-terminal segment of each protein, called thevariable (V) region, is composed of noncollagenous sequence and consistsof 67 amino acids in Scl1.1 and 73 amino residues in Scl2.28.

To examine the structural organization of Scl1, three recombinantproteins, P144 (SEQ ID No: 47), P157 (SEQ ID NO: 48), and P158 (SEQ IDNO: 49), were constructed representing the combined VCL regions, the CLregion, and the V region, respectively (FIG. 1A). Wild typestreptococcal Scl1.1 migrates aberrantly in denaturing SDS-PAGE.Similarly, the recombinant P144 and P157 also migrates aberrantlywhereas P158 did not (FIG. 1B, left). Therefore, the recombinant proteinidentities were confirmed by amino-terminal sequencing and their massesof 26.8, 18.5, and 9.4 kDa, respectively, were verified by massspectrometry analyses.

Furthermore, both P144 and P158 reacted with the V region-specificantibodies, whereas P157 did not (FIG. 1B, right). A recombinant proteinP163 containing the combined VCL regions of the Scl2.28 was generatedand purified. Recombinant P163 contains 79 GXY repeats in thecollagen-like region and 72 amino acids in the V region. P163(M_(r)=33.8) also migrated aberrantly in SDS-PAGE and its identity wasconfirmed by amino-terminal sequencing.

The recombinant P176 protein has an 84 amino-acid long V region followedby a CL region that includes 62 GXY triplets. It adopted atriple-helical conformation and formed lollipop-like structures whenviewed by EM (FIG. 1C). Both P163 and P176 were affinity purified toapparent homogeneity, as seen by the presence of single bands onSDS-PAGE (FIG. 1D). The integrity of each protein sample was furtherverified by western immunoblotting with specific polyclonal antibodies(data not shown).

EXAMPLE 3

Multimolecular Organization of Scl Proteins

To assess the ability of Scl1 and Scl2 to assemble into polymericstructures, the electrophoretic mobilities of P144, P157, and P163 inpolyacrylamide gel run in the absence of SDS before and after heatdenaturation were compared (FIG. 2A). In each case the native samplesproduced uniform higher molecular mass bands compared with theircorresponding samples that were heat-denatured before loading on a gel,results suggesting that all three proteins tested formed oligomers undernondenaturing conditions. The presence of multiple bands in denaturedP144 and P163 suggested that these proteins partially renatured whilerunning on a gel.

In subsequent experiments, the mobility of native and heat-denaturedsamples of P144 and P163 were compared in SDS-PAGE using molecular massstandards (FIG. 2B). A heat-denatured P144 (M_(r)=26.8) migrated as a45-kDa band, whereas the nondenatured sample appeared as a single bandwith an apparent M_(r) of 140–160 kDa. Similarly, the heat-denaturedP163 migrated as a single band of ˜43 kDa, whereas nondenatured P 163appeared heterogeneous with a predominant band that migrated in therange of 130–140 kDa. Hence, native Scl proteins form higher orderedstructures and gel mobility data suggested that P144 form a stabletrimer under the SDS-PAGE conditions used. Also P163 appears to form atrimer, but this putative trimer may partly dissociate in SDS-PAGE.

To test the formation of triple helix by Scl proteins, thesusceptibility of recombinant P144 to trypsin was examined (FIG. 2C).Because triple helix is resistant to trypsin, the collagen-like domainof P144 should be protected against digestion. Native P144 was quicklytrimmed to the size of the collagen-like domain corresponding to P157and then remained relatively resistant to proteolysis. In contrast, thedenatured P144 was nearly completely degraded within 15 min of theexperiment. These results strongly suggested that CL region in Sclproteins is triple helical.

EXAMPLE 4

Circular Dichroism (CD) Spectroscopy of Recombinant Scl Proteins

Collagen triple helices have characteristic CD spectra with a positiveellipticity maximum at 220 μm. To examine the secondary structurecomposition of Scl1 and Scl2, recombinant proteins were analyzed by CDspectroscopy (FIGS. 3A–3C, solid lines). The CD spectrum of P157 at 25°C. resembled that of a collagen triple helix, with an ellipticitymaximum at 220 nm in the order of 1×10³ (degree cm² dmol¹) (FIG. 3B).Similarly, the CD spectrum of P144 corresponding to the combined VCLregions also included the characteristic shoulder at 220 nm (FIG. 3A).

The peak of the shoulder, however, was of a negative value probably dueto the contribution of the secondary structure of the noncollagenous Vdomain. Similarly small amplitudes were previously reported for somemammalian proteins with collagenous domains including surfactantproteins and complement component C1q. The CD spectrum of the Scl2.28,recombinant P163, also had the characteristics of a collagen triplehelix with ellipticity maximum at 220 nm (˜2×10³ degree cm² dmol¹) (FIG.3C).

The CD spectrum of P158 (10 μM) did not include the characteristics of acollagen triple helix (FIG. 3G). Deconvolution of the spectrum indicatedthat the recombinant V domain consisted of 25.7% (±4.3%) a-helices, 43.6(±10.5%) b-sheets, and 30.7% (±11.3%) other secondary structureelements.

When samples of P144 (8.1 μM), P157 (29.8 μM) and P163 (7.7 μM) wereheated to 50° C., the CD spectra recorded at this temperature (FIGS.3A–3C, broken lines) did not show the characteristic features of acollagen triple helix, e.g., the 220 nm maximum, but rather indicated arandom coil structure, suggesting that the triple helix had unfolded. Bymonitoring the CD at 220 nm as a function of increasing temperature, thethermal unfolding of the Scl proteins were followed. The triple-helicalstructure in all three proteins unfolded within a very narrowtemperature range (t<=6° C.), with the midpoint temperatures oft_(m)=36.4±0.4° C. for P144 (FIG. 3D), t_(m)=37.7±0.2° C. for P157 (FIG.3E), and t_(m)=37.6±0.8° C. for P163 (FIG. 3F). The sharp transition andthe t_(m) values of these proteins are reminiscent of the transitionfrom triple helix to random coil seen for a type I collagen, i.e.t_(m)=38° C. and t=3° C.

The thermal unfolding of the collagen-like triple helix of P144 and P163appears to be readily reversible. When the protein samples were cooled,the ellipticity at 220 nm gradually increased (FIGS. 3D–3F), and the CDspectra of samples cooled to 4° C. again showed the characteristics of acollagen-like triple helix (FIGS. 3A and 3C, dotted lines). The signalintensities at 207 and 220 nm of the refolded proteins were lower thanthose recorded for the original proteins before melting, suggesting thatthe refolding was not complete. The attempts to refold P157 wereunsuccessful, suggesting that the amino-terminal V regions present inP144 and P163 but absent in P157 might facilitate triple helix assembly.The above experiments were also performed with a slower temperatureslope (10° C./h), and no obvious differences were found.

The CD spectra of P176 at 4° C. and 25° C. showed ellipticity maxima at220 nm, which is consistent with a collagen triple helix-like structure(FIG. 3H). When the P176 sample was heated to 50° C., the CD spectrum nolonger exhibited the characteristics of a triple helix, but ratherindicated a random coil structure, suggesting that the triple helix hadunfolded. Upon cooling to 4° C. again, the signal intensities at 220 nmhad increased, indicating that the triple helix could reassemble.

EXAMPLE 5

Electron Microscopy of Scl Proteins

Electron microscopy of rotary shadowed samples has been used forstudying the structural organization of collagens and collagen-likemammalian proteins. Examination of a rotary shadowed preparation ofScl1, e.g., recombinant P144, revealed a two-domain lollipop-likestructure (FIGS. 4A–4B) and ˜25% of the molecules appeared to formdimers via head-to-head interactions (FIGS. 4A and 4C). It iscontemplated that the collagen-like region of Scl1 formed the lollipopstalk, whereas the globular head was made of the V region. Two lines ofevidence support this hypothesis. First, the recombinant P157 proteincorresponded to the CL region formed rod-shaped particles (FIG. 4D) and,secondly, the anti-V specific antibody appeared to bind to the globularheads of Scl1 (FIG. 4E). Scl2, e.g., recombinant P163, also had atwo-domain lollipop-like structure (FIG. 4F); however, in contrast toScl1 preparation, Scl2 did not appear to form head-to-head dimers.

The measured contour length, i.e., 279 particles measured, of thecollagenous tail in P144 is 45.5±4.3 nm. Based on a translation of 0.286nm per residue observed in a collagen triple helix, the 150-residuestretch encompassing 50 GXY repeats in the CL region was calculated tobe 43 nm long, which correlated well with the measured length.Rod-shaped particles (n=190) of similar length 45.6±4.4 nm were seen inthe P157 preparation, further confirming that the CL region of Scl aloneformed a triple helix.

The shape of the globular domain in P144 was more heterogenous with somemolecules appearing spherical and others elongated extending along theaxis of the collagenous tail. The diameter of the globular domain(n=279) measured in the middle of the head perpendicularly to the axisof the tail was 8.9±2.2 nm. Considering size overestimation by 2.5 to 5nm due to metal decoration, a diameter of 3.9–6.4 nm appeared morerealistic. The latter estimate was close to the theoretical value of 3.8nm calculated for a spherically shaped protein with a molecular mass ofa trimeric V region using the equation: d=²(3vM_(r)/4πN_(A))/^(1/3),where M_(r)=25,072, N_(A) is Avogadro's number, and v=0.73 cm³/g is theassumed partial specific volume.

The dimensions of P163 also agreed with theoretical predictionscalculated for the Scl2.28 protein. Specifically, the measured length ofthe collagenous domain (n=253) of 66.2±8.9 nm correlated well with thecalculated value of 67.8 nm. Similarly, a diameter of the globulardomain (n=124) of 7.4±0.9 nm agreed with the calculated value of 3.9 nm,considering a metal decoration component. Examination of a rotaryshadowed preparation of P176 (FIG. 4G) revealed a characteristictwo-domain lollipop-like structure, consistent with what has beenpreviously reported for other Scl variants, such P163.

EXAMPLE 6

Computer Modeling

The trimeric structure of the collagen-like region of Scl1.41 andScl2.28 was homology-modeled using SwissModel, based on the coordinatesof a (Pro-Pro-Gly)₁₀ collagen-like peptide. The target-templatealignment was guided by superposition of Gly residues. Energyminimization in vacuo was carried out using GROMOS96 to relieve shortcontacts and improve local stereochemistry. Interactions involving theside chains have not been modeled explicitly.

The amino acid sequence of the collagenous domains of Scl1.41 andScl2.28 was fitted onto the structure of a regular polyGPP triple helix(FIG. 5A). All steric clashes between the polypeptide backbone and thenewly introduced side chains were relieved by energy minimizationwithout significant alteration of the main chain torsion angles.Moreover, the lengths of the Scl1.1CL and Scl2.28CL models, 42.3 nm and67.1 nm, respectively, agreed well with the experimentally determinedlengths of these regions, as determined in Example 5, providing furtherevidence supporting the validity of the models.

Although the CL regions in Scl1.41 and Scl2.28 are both composed ofcontinuous GXY repeats, they show remarkable primary sequencedifferences. The five most frequent triplets in Scl1.1 GEK, GPQ, GEA,GET, and GPA, account for 48% of all triplets in this protein butrepresent only 10% of the triplets in Scl2.28. Similarly, the GKD, GAQ,GPA, GER, and GLP triplets make up 55.9% of the collagen-like segment inScl2.28 but account for only 16% of the triplets in Scl1.1. Fifty GXYmotifs of Scl 1. CL contain 21 distinct triplets, seven of which are notfound in Scl2.28. Conversely, 12 out of 26 distinct triplets arespecific to Scl2.28CL.

In addition, those GXY motifs that are common to both Scl proteins arearranged in different orders in Scl1.1CL and Scl2.28CL. Despite thesignificant sequence variation in the CL region of the two proteins,these could both successfully be modeled on a polyGPP structure. Thusthe ability to form a collagen-like triple helix structure is not uniqueto a particular Scl sequence but may be the property of otherprokaryotic proteins containing repeated GXY motifs.

EXAMPLE 7

Collagen-like Protein Promotes Cell Adhesion and Spreading

The expression levels of various integrin chains on MRC-5 and WI-38cells were determined using fluorescence activated cell sorting (FACS).Both cell types expressed collagen binding α₁β₁ and α₂β₁ integrins atcomparable levels, as well as α₃β₁, α₅β₁ and α_(v1) which presumablypartners with β₅ (FIGS. 6A–6B).

Next, cell adhesion assays were performed on immobilized recombinantP1163 and P176 proteins or known adhesive extracellular matrix (ECM)proteins. P176 served as a substrate for the attachment of both MRC-5and WI-38 cells, similar to fibronectin (FN), type IV collagen (Col IV),and type I collagen (Col I). The extent of cell attachment to thesesubstrates depended on the amount of protein used to coat the wells andthe incubation time of the cells (FIGS. 6C–6D). In contrast, P163 didnot support cell adhesion under the same experimental conditions.

The cell attachment results were complemented with cell spreadingassays. Detached WI-38 and MRC-5 cells were allowed to reattach ontodishes coated with FN, Col I and P176 substrates for 45 or 90 minutes.P176 induced considerable cell spreading, as did the positive controladhesion substrates (FIGS. 7A–7B). Under the same conditions, cellattachment to P163 was negligible and the few cells that did attach didnot undergo cell spreading, but were removed by gentle washing (data notshown).

Cell spreading was further examined by evaluating cell morphology influorescent-stained MRC-5 cells (FIG. 3C). Cells incubated on substratesof P176, Fn or Col I adopted a morphology characteristics of fullyspread fibroblasts with a defined nucleus (DAPI stained) surrounded byan extended cytoplasm. The assembly of actin filaments into stressfibers was demonstrated in cells stained with tetramethylrhodamine Bisothiocyanate (TRITC)-phalloidin. Furthermore, when MRC-5 cells wereincubated on P176 substrates for a prolonged period (˜2–3 hours), theyexhibited an elongated and contractile appearance, similar to thesecells incubated on Col I (data not shown). These results show that Sclprotein interacts with integrin(s) to induce cell attachment and promotecell spreading.

EXAMPLE 8

Cell Adhesion Activity of P176 is Mediated via Its Collagen-like Region

To delineate the regions of the P176 protein responsible for its abilityto support cell adhesion and spreading activity, two chimericrecombinant proteins, P181 (SEQ ID NO: 50), and P182 (SEQ ID NO: 51),were generated by exchanging domains between the putativeintegrin-binding variant P176 and the non-interacting variant P163(FIGS. 8A and 8D). The V region of P163 was replaced with thecorresponding P176-V region to create P 181. The P 176-CL region wasthen substituted for the corresponding CL region of P181, originally aP163-CL sequence, resulting in P182. Neither of the chimeric proteinscontained the repeats of the linker region found in P176. Analyses ofpurified recombinant P181 and P182 proteins by electron microscopy andfar UV CD spectroscopy suggest that both chimeric proteins behaved asScl proteins (41).

Both P181 and P182 were able to form collagen-like triple helices, asdetermined by CD (FIGS. 8B and 8E) and electron microscopy (FIGS. 8C and8F). Cell adhesion assays showed that only P182 which contains theP176-CL region, but not P181 which contains P176-V and P163-CL domains,supported cell adhesion activity in a time- and dose-dependent manner inboth the MRC-5 and WI-38 cell lines (FIGS. 8G–8H). This datademonstrates that the collagenous CL region, but not the globular V,region of P176 supports cell adhesion, possibly through interacting withone or more of the collagen-binding integrins.

EXAMPLE 9

P176 Interacts Specifically with the α₁β₁ Collagen-binding Integrin

To determine which collagen-binding integrin(s) recognizes P176, wetried to inhibit cell attachment using a panel of monoclonal antibodiesdirected against the extracellular segments of human integrins. Theability of MRC-5 cells to attach onto plates coated with P176 wasmeasured in the presence of increasing concentrations of anti-α₁β₁,-α₂β₁, -α₃β₁, -α_(v)β₃, -α₅β₁, or -β₁ integrins adhesion-blockingmonoclonal antibodies, or in the presence of EDTA, a metal ion chelatingagent (FIG. 9A). Interestingly, only anti-a₂β₁ and −β₁ integrinantibodies inhibited cell adhesion activities in a dose dependentmanner. In contrast, antibodies against other integrins, including thoseagainst the integrins α₁β₁, α₃β₁, α₅β₁ and α_(v)β₃ did not block cellattachment. As expected, EDTA also inhibited cell attachment in adose-dependent manner. These data show that α₂β₁ integrin is a cellularreceptor for the P176 protein.

To further examine the role of the α₂β₁ integrin in cell adhesion toP176, collagen receptor deficient C2C12 myoblast cells, and C2C12 cellsstably expressing the human wild-type α₂ integrin subunit (designatedC2C12-α₂+) were used. Upon expression in C2C12 cells, wild-type₂polypeptide combines with the endogenous β₁ subunit, to form afunctional α₂β₁ integrin (24). Col I, Col IV, FN, and VN were includedas positive controls in cell adhesion assays. As expected, Col I and ColIV failed to promote adhesion of parental C2C12 cells, in contrast, bothECM proteins supported adhesion of C2C12-α₂+ cells (FIG. 9B). Consistentwith previous report, FN and VN supported attachment of both C2C12parental and C2C12-α₂+ cells (FIG. 9B).

If C2C12-α₂+ adheres to P176 through integrins(s), does adhesionblocking anti-integrin antibodies inhibit their interactions? Theattachment of both parental and C2C12-α₂+ cells to BSA (negativecontrol) and P163 were insignificant (FIG. 9C). Similarly, adhesion ofparental C2C12 cells to P176 was also negligible (FIG. 9C). In contrast,C2C12-α₂+ cells attached to P176 protein productively and thisinteraction was blocked effectively by anti-α₂β₁ and anti-₁ integrinmonoclonal antibodies (FIG. 9C). Clearly, these data indicate thatadhesion of cells to P176 is mediated specifically by α₂b₁ integrin.

These findings further support the hypothesis that cell adhesion ismediated through α₂β₁. Furthermore, because P176 interacts directly withα₂β₁ integrin, the data may support a caveolae-mediated entry route forcertain S. pyogenes strains. Both the paracellular and the caveolarmechanisms may be advantageous for the propagation of GAS and for itspathogenicity.

EXAMPLE 10

The I-Domain of α₂ Integrin (α₂-I) Interacts with P176 but not P181

The I-domain of the a components of collagen binding integrins have beendemonstrated to directly bind to specific site in triple helix collagens(62). To study if the I-domain of a₂ integrin subunit binds to P176,α₂-I was expressed as a recombinant protein and its binding to P176 andP181 examined by surface plasmon resonance (SPR) spectroscopy. Differentconcentrations of α₂-I were passed over Bia-core chips to which therecombinant Scl proteins had been coupled.

A concentration dependent binding of α₂-I to P176 was noted with rapidon and off rates and defined equilibrium (FIG. 10A). A K_(D) of 17 nMwas calculated from a Scatchard plot of the equilibrium data (FIG. 10B).No significant binding was observed when increasing concentrations (upto 300 nM) of a₂-I were run over P181 (FIG. 10C). These resultsdemonstrate that P176 contains a site to which α2-I domain binds withhigh affinity.

EXAMPLE 11

P176 Induces Tyrosine Phosphorylation of p125FAK, p130CAS, Paxillin, andJNK

Cell adhesion-induced tyrosine phosphorylation of p125FAK, p130CAS,paxillin proteins and JNK are considered to be integrin-mediatedsignaling events (36). The phosphorylation state of these proteins wasevaluated to determine whether P176 induces α₂β₁ integrin signaling inthis manner. Cells kept in suspension were used as a negative controland cells adhering to Col I and FN substrates were used as positivecontrols.

Cells were incubated with the various substrates for 30 min, a timerequired for FAK activation, and the lysates were analysed as shown inFIGS. 11A–11H. FIG. 11A shows that the phosphorylation of p125FAK attyrosine-397 is markedly increased in response to adhesion onto P176,FN, and Col I. Since paxillin and p130CAS proteins are key focaladhesion-related molecules that undergo phosphorylation in response toactivation of p125FAK, the tyrosine phosphorylation states of theseproteins also was examined.

Immune complexes were analyzed by immunoblotting with ananti-phospho-tyrosine antibody. Like p125FAK, both p130CAS and paxillinwere phosphorylated in response to adhesion onto P176, FN, and Col I(FIGS. 11C and 11E). In contrast, these proteins did not exhibit anychange in tyrosine phosphorylation state in the cells that remained insuspension, mimicking the unattached cells on P163.

Similarly, phospho-JNK immunoblotting analysis of total lysates showedthat JNK was phosphorylated in response to adhesion onto P176, FN andCol I (FIG. 11G), but not under control conditions (cells insuspension). All blots were stripped and reprobed with antibodiesdirected against the corresponding proteins, or a non-phospho-specificantibody in the case of FAK and JNK, to verify that equivalent amountsof proteins were loaded in all the samples (FIGS. 11B, 11D, 11F, and11H). The data indicate that a₂b₁ integrin dependent adhesion to P176induces phosphorylation of FAK, CAS, Paxillin and JNK proteins.

The data indicate that fibroblast cells attach and spread on P176 andthat these events are associated with the formation of focal adhesioncontacts where vinculin and talin localize. P176-mediated integrinclustering and signaling is consistent with the time-dependentactiviation of FAK and JNK protein kinases observed in fibroblastsplated on P176. In fibroblast and endothelial cells, p38 MAP kinase andATF-2 are activated in response to α₂β₁ integrin ligation (67–68). ThusP176 promotes tyrosine phosphorylation of integrin signaling moleculesin fibroblasts. This is consistent with the hypothesis that Scl1.41 is abacterial collagen that induces signaling through the cell adhesionreceptor α₂β₁ integrin.

The following references are cited herein:

-   1. Prockop, D. J. 1998. Matrix Biol. 16:519–528.-   2. Ramachandran, G. N. 1988. Int. J. Pept. Protein. Res. 31:1–16.-   3. Brodsky, B., and N. K. Shah. 1995. FASEB J. 9:1537–1546.-   4. Brodsky, B., and J. A. Ramshaw. 1997. Biol. 15: 545–554.-   5. Uitto, et al., 1973. Biochem. Biophys. Res. Comm. 55: 904–911.-   6. Uitto, J., and D. J. Prockop, 1974. Biochemistry. 22:4586–4591.-   7. Pihlajaniemi et al., 1981. Biochemistry. 20:7409–7415.-   8. Leblond, C. P. 1989. Anat. Rec. 224:123–138.-   9. Berisio, et al., 2002. Protein Sci. 11:262–270.-   10. Sellar, et al., 1991. Biochem. J. 274:481–490.-   11. Reid, K. B. M. 1993. Biochem. Soc. Trans. 21:464–468.-   12. Engel, J. 1997. Science. 277:1785–1786.-   13. Charalambous, et al., 1988. EMBO J. 7:2903–2909.-   14. Smith, et al., 1998. Science. 279:834.-   15. Ferretti, et al., 2001. Proc. Natl. Acad. Sci. USA.    98:4658–4663.-   16. Rasmussen, et al., 2003. J. Biol. Chem. 278:32313–32316.-   17. Hynes, R. O. 1987. Cell. 27:549–554.-   18. Santoro, et al., 1995. Thromb. Haemost. 74:813–821.-   19. Ruoslahti, E. 1991. Integrins. J. Clin. Invest. 87:1–5.-   20. Albelda, S. M., and C. A. Buck. 1990. FASEB J. 11: 2868–2880.-   21 Cheresh, D. A. 1992. Clin. Lab. Med. 12:217–236.-   22. Larson, et al., 1990. Immunol. Rev. 114:181–217.-   23. Newham, et al., 1996. Mol. Med. Today. 2:304–313.-   24. Tiger, et al., 2001. Dev. Biol. 237:116–129.-   25. Velling, et al., 1999. J. Biol. Chem. 274:25735–25742.-   26. Camper, et al., 2001. Cell Tissue Res. 306:107–116.-   27. Kern, et al., 1993. Eur. J. Biochem. 215:151–159.-   28. Kern, et al., 1994. J. Biol. Chem. 269:22811–22816.-   29. Kern, et al., 1998. J. Cell Physiol. 176:634–641.-   30. Nykvist, et al., 2000. J. Biol. Chem. 275:8255–8261.-   31. Xiong, et al., 2003. J. Thromb. Haemost. 1:1642–1654.-   32. Liddington, et al., 1998. Structure. 6:937–938.-   33. Heino, J. 2000. Matrix Biol. 19:319–323.-   34. Gullberg, et al., 2002. Prog. Histochem. Cytochem. 37:3–54.-   35. Yamada, K. M. 1997. Matrix Biol. 16:137–141.-   36. Giancotti, et al., 1999. Science. 285:1028–1032.-   37. Stupack, et al., 2002. J. Cell. Sci. 115:3729–3738.-   38. Martin, et al., 2002. Science. 296:1652–1653.-   39. Miranti, C. K., and J. S. Brugge. 2002. Nat. Cell Biol.    4:E83–90.-   40. Alahari, et al., 2002. Int. Rev. Cytol. 220:145–184.-   41. Lukomski, et al., 2000. Infect. Immun. 68:6542–6553.-   42. Lukomski, et al., 2001. Infect. Immun. 69:1729–1738.-   43. Rasmussen, et al., 2000. Infect. Immun. 68:6370–6377.-   44. Rasmussen, et al., 2001. Infect. Immun. 40:1427–1438.-   45. Whatmore, A. M. 2001. Microbiology. 147:419–429.-   46. Okada, et al., 1995. Proc. Natl. Acad. Sci. USA. 92:2489–2493.-   47. Schrager, et al., 1998. J. Clin. Invest. 101:1708–1716.-   48. Stockbauer et al., 1999. Proc. Natl. Acad. Sci. USA. 96:242–247.-   49. Cue, et al., 1998. Infect. Immun. 66:4593–4601.-   50. Cywes, et al., 2001. Nature. 414:648–652.-   51. Patti, et al., 1994. Curr. Opin. Cell Biol. 6:752–758.-   52. Ozeri, et al., 1998. Mol. Microbiol. 30:625–637.-   53. Rohde, et al., 2003. Cell. Microbiol. 5:323–342.-   54. Glenney et al., 1992, Proc. Natl. Acad. Sci. USA.    89:10517–10521.-   55. Rothberg, et al., 1992. Cell. 68:673–682.-   56. Wary, et al., 1998. Cell. 94:625–634.-   57. Wary, et al., 1996. Cell. 87:733–743.-   58. Wei, et al., 1996. Science. 273:1551–1555.-   59. Kagawa, et al., 2000. Proc. Natl. Acad. Sci. USA. 97:2235–2240.-   60. Molinari, et al., 1997. Infect. Immun. 65:1357–1363.-   61. Cue, et al., 2000. Proc. Natl. Acad. Sci. USA. 97: 2858–2863.-   62. Xu, et al., 2000. J. Biol. Chem. 275:38981–38989.-   63. Sakai, et al., 1994. Methods Enzymol. 245:29–52.-   64. Humtsoe, et al., 2003. EMBO J. 22:1539–1554.-   65. Rich, et al., 1999. J. Biol. Chem. 274:24906–24913.-   66. Wary, et al., 1999. Methods Mol. Biol. 129:3549.-   67. Xu, et al., 2001. Biochem. J. 355:437–447.-   68. Ivaska, 1999. J. Cell Biol. 147:401–416.

Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. Further, these patents and publications areincorporated by reference herein to the same extent as if eachindividual publication was specifically and individually incorporated byreference.

One skilled in the art will appreciate readily that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those objects, ends and advantagesinherent herein. Changes therein and other uses which are encompassedwithin the spirit of the invention as defined by the scope of the claimswill occur to those skilled in the art.

1. A recombinant collagen-like protein comprising one or more domainshaving one or more of the amino acid sequences of SEQ ID NOS: 16–18, 20,21 and 23–25, wherein said protein thereby forms a triple helicalstructure.
 2. The recombinant collagen-like protein of claim 1, furthercomprising one or more triple helical domains of a mammalian collagen.3. The recombinant collagen-like protein of claim 2, wherein saidmammalian collagen is human collagen.
 4. The recombinant collagen-likeprotein of claim 2, wherein the one or more amino acid sequences havingSEQ ID NOS: 16–18, 20, 21 and 23–25 are fused to said one or more triplehelical domains of mammalian collagen.
 5. A chimeric collagen-likeprotein comprising one or more domains having one or more of the aminoacid sequences of SEQ ID NOS: 16–18, 20, 21 and 23–25; and one or moretriple helical domains of a mammalian collagen or peptide therewithinhaving a triple helical structure.
 6. The collagen-like protein of claim5, wherein said mammalian collagen is human collagen.
 7. The chimericcollagen-like protein of claim 5, wherein said protein is a syntheticprotein or a fusion protein.
 8. A recombinant collagen-like proteinconsisting of one or more domains which consists of one or more of theamino acid sequences of SEQ ID NOS: 19 or 22, wherein said proteinthereby forms a triple helical structure.
 9. The recombinantcollagen-like protein of claim 8, further comprising one or more triplehelical domains of a mammalian collagen.
 10. The recombinantcollagen-like protein of claim 9, wherein said mammalian collagen ishuman collagen.
 11. The recombinant collagen-like protein of claim 9,wherein the one or more amino acid sequences consisting of SEQ ID NOS:19 or 22 are fused to said one or more triple helical domains ofmammalian collagen.
 12. A chimeric collagen-like protein consisting ofone or more domains which consists of one or more of the amino acidsequences of SEQ ID NOS: 19 or 22; and one or more triple helicaldomains of a mammalian collagen or peptide therewithin having a triplehelical structure.
 13. The collagen-like protein of claim 12, whereinsaid mammalian collagen is human collagen.
 14. The chimericcollagen-like protein of claim 12, wherein said protein is a syntheticprotein or a fusion protein.